1. Technical Field of the Invention
The present invention relates to methods for detecting antisense oligonucleotides and peptide nucleic acids which are labeled with stable isotopes, and foreign antisense chains.
2. Background of the Invention
In recent years, antisense drugs using antisense technology have drawn attention as therapeutic agents for diseases such as cancers, genetic diseases and AIDS. An antisense drug refers to an oligonucleotide or the like that has a sequence complementary (antisense) to a part of a sequence of a specific gene which causes a certain disease. When introduced into the body (target cells), the antisense drug forms a specific double strand chain with mRNA, which is a transcription product of the causative gene, or a precursor thereof, to inhibit translation or processing of the precursor mRNA. Thus, this inhibition can control the onset of the disease. The efficacy of antisense drugs heavily depends on the technique to deliver the oligonucleotides to the target site. This is because nucleases that decompose nucleic acids are generally present in the body and digest the introduced oligonucleotides before they reach the target site, which disables the formation of the complementary double strand chains at the target site, thus no effect can be obtained. For example, a nucleic acid incorporated into a cell by endocytosis is incorporated into a lysosome in the cell, and then decomposed by a nuclease in the lysosome. Thus, the introduced oligonucleotide cannot form a double strand chain with the target transcription product in the nucleus or cytoplasm, resulting in no effect. An example of a useful delivery technique to solve this problem is the use of intracellular routing agents, typically represented by a liposome preparation, which has provided a certain level of success.
Another attempt to improve the effect of antisense drugs involves increasing the stability of the antisense chains by modifying the nucleotides, the structural units of oligonucleotides, to non-natural modified nucleotides which are less susceptible to decomposition by nucleases.
In developing therapeutic drugs, pharmacokinetic tests for absorption, metabolism and excretion of the drugs are carried out. In pharmacokinetic tests, substances to be tested (drugs) are labeled with radioactive isotopes or the like and administered to experimental animals, and the concentration and radioactivity of the drugs are quantitatively measured with the lapse of time. Antisense drugs are no exception and antisense oligonucleotides labeled with radioactive isotopes or the like are subjected to pharmacokinetic tests.
In the use of antisense oligonucleotide drugs, the sequence and length of the nucleotides have to be conserved in body cells to fully implement their function. However, conventional pharmacokinetic tests using radioisotopes or the like provide information on the distribution in the body, absorption rate, and excretion rate of the oligonucleotides but not on the conservation of their sequence and length. Accordingly, the state of the conservation of oligonucleotides has to be confirmed by extracting a nucleic acid fraction from blood, organs or the like taken from experimental animals to analyze oligonucleotides in the fraction by high performance liquid chromatography, Southern blotting, Northern blotting, capillary electrophoresis, or the like. Furthermore, it was virtually impossible to see the change in the length of the administered antisense nucleotides with the lapse of time (the progress of decomposition). Further, there is no means to confirm whether the target mRNA or precursor mRNA and the oligonucleotide introduced into cells form complementary double strand chains. Moreover, the efficacy of the introduction of the oligonucleotide can only be judged cytologically based on physiological or morphological changes of the cells to which the oligonucleotide is introduced. Furthermore, in conventional pharmacokinetic tests, the use of radioisotopes is inevitable, which requires a special facility according to specified regulations and well-trained technicians.